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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Circ Res. Author manuscript; available in PMC Jun 22, 2013.
Published in final edited form as:
PMCID: PMC3390172
NIHMSID: NIHMS386108
Inefficient reprogramming of fibroblasts into cardiomyocytes using Gata4, Mef2c, Tbx5
J.X. Chen,1* M. Krane,1,2* M. A. Deutsch,1,2 L. Wang,3 M. Rav-Acha,1 S. Gregoire,1 M. C. Engels,1 K. Rajarajan,1 R. Karra,4 E. D. Abel,5 J. C. Wu,3 D. Milan,1 and S. M. Wu1,6
1Cardiovascular Research Center, Division of Cardiology, Department of Medicine, Massachusetts General Hospital, Boston, MA
2German Heart Center Munich, Department of Cardiovascular Surgery, Department of Experimental Surgery, Technische Universität München
3Division of Cardiovascular Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, CA
4Division of Cardiology, Department of Medicine, Duke University School of Medicine, Durham, NC
5Division of Endocrinology, Metabolism and Diabetes, and Program in Molecular Medicine, University of Utah School of Medicine, Salt Lake City, UT
6Harvard Stem Cell Institute, Cambridge, MA
Corresponding author: Sean M. Wu, MD, PhD, Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, 185 Cambridge Street, Boston, MA 02114, Phone: 617-643-3458, Fax: 617-643-3451, smwu/at/partners.org
*Contributed equally to this work
Rationale
Direct reprogramming of fibroblasts into cardiomyocytes is a novel strategy for cardiac regeneration. However, the key determinants involved in this process are unknown.
Objective
To assess the efficiency of direct fibroblast reprogramming via viral overexpression of GATA4, Mef2c, and Tbx5 (GMT).
Methods and Results
We induced GMT overexpression in murine tail tip fibroblasts (TTFs) and cardiac fibroblasts (CFs) from multiple lines of transgenic mice carrying different cardiomyocyte lineage reporters. We found that the induction of GMT overexpression in TTFs and CFs is inefficient at inducing molecular and electrophysiological phenotypes of mature cardiomyocytes. In addition, transplantation of GMT infected CFs into injured mouse hearts resulted in decreased cell survival with minimal induction of cardiomyocyte genes.
Conclusions
Significant challenges remain in our ability to convert fibroblasts into cardiomyocyte-like cells and a greater understanding of cardiovascular epigenetics is needed to increase the translational potential of this strategy.
Keywords: gene expression, Ca++ channels, cardiac development, myocardial ischemia, myocyte regeneration
Unlike the hearts of teleosts or zebrafish,1 the mammalian heart undergoes a fibrotic response with minimal regeneration.2 The inability of the adult heart to completely repair itself spurs the development of strategies to transplant endogenously or exogenously-derived cardiac cells into patients after ischemic injury38. These transplantation strategies have led to measurable successes in functional recovery or remuscularization. However, significant challenges remain regarding the efficiency, feasibility and efficacy of this approach69. Thus, strategies aimed at directly converting cardiac scar fibroblasts into cardiomyocytes are appealing as they circumvent problems associated with cell purity and survival after transplantation.
To this end, Ieda and colleagues reported that overexpression of Gata4, Mef2c and Tbx5 (GMT) could reprogram murine cardiac fibroblasts (CFs) and tail tip fibroblasts (TTFs) into cardiomyocytes in vitro.10 Furthermore, infected fibroblasts could survive and reprogram after transplantation into a murine heart. GMT-induced fibroblasts demonstrated gene expression profiles similar to mature cardiomyocytes and beat spontaneously in vitro. Conversion to cardiomyocyte-like epigenetic states was reported through the de-repression of histone markings at promoters of sarcomeric genes. These results implicated therapies that can directly remuscularize the heart without the need for cell transplantation, provided that the efficiency of reprogramming is sufficiently robust.
Here, we evaluated the efficiency of this direct cardiac reprogramming strategy using the GMT expression viruses reported in Ieda et al.10 and validated myocardial lineage reporters (αMHC-Cre, Nkx2.5-Cre, cTnT-Cre). We found a lack of αMHC or Nkx2.5 reporter activation in CFs and TTFs despite significant overexpression of GMT factors. However, with cTnT reporter we observed a ~35% labeling of fibroblasts that is confirmed by a ~250-fold increase in cTnT expression. However, the expression of other cardiac genes was minimally elevated. With GMT infection, we found 22% of infected fibroblasts exhibited a voltage-dependent calcium current without a spontaneous action potential, suggesting incomplete electrophysiological reprogramming. Furthermore, GMT-infected fibroblasts exhibited poor survival and minimal cardiac gene expression following transplantation into an injured murine heart in vivo. Together, our data suggest that direct cardiomyocyte reprogramming by GMT factors is inefficient and a greater understanding of transcription factor-mediated epigenetic change will be need to translate this promising approach into therapy.
Methods
The lentiviral tetracycline-inducible GMT expression vectors reported in Ieda et al.10 were kindly provided by Dr. Deepak Srivastava.
Detailed methods can be found in the Supplemental Materials.
Reprogramming tail tip fibroblasts by GMT overexpression
To assess the efficiency of cardiomyocyte reprogramming from TTFs, we used αMHC-Cre/ROSA26mTmG mice that express membrane-tethered tandem dimerized Tomato (dTomato) at baseline, and switch to membrane-tethered enhanced GFP (eGFP) upon Cre-mediated excision in the ROSA locus (ROSA26mTmG) (Figure 1a,b)11. αMHC-Cre/ROSA26mTmG hearts are eGFP+ (Figure 1c) but TTFs are dTomato+ prior to GMT overexpression (Figure 1d). We infected freshly-isolated TTFs from αMHC-Cre/ROSA26mTmG mice with lentiviruses constitutively expressing rtTA along with doxycycline-inducible lentiviruses expressing Gata4, Mef2c, and Tbx5. Following induction with doxycycline for three weeks, we found no eGFP+ cells by immunofluorescence microscopy and flow cytometry with or without GMT lentiviral infection (Figure 1d).
Figure 1
Figure 1
Lentiviral-mediated overexpression of Gata4/MEF2C/Tbx5 factors in CFs and TTFs
To investigate this unexpected finding, we evaluated the induction of GMT overexpression in infected TTFs and found up to 1000-fold increases in GMT factors (Online Figure Ia). Although upregulation of Mef2c in infected TTFs remained modest (~10-fold) despite increases in viral titer, further investigation revealed that baseline levels of Mef2c in uninfected TTFs are already significantly elevated (Online Figure Id). Immunocytochemical staining for GMT proteins demonstrated their nuclear localization (Online Figure Ib). Luciferase reporter assays using enhancer/promoter elements previously described to report the transcriptional activities of Gata4, Mef2c, and Tbx5 proteins1214 confirmed that each transcription factor is active in vitro (Online Figure Ic). To ensure that the ROSA26mTmG reporter can be efficiently excised by αMHC-Cre in vitro, we generated and differentiated αMHC-Cre/ROSA26mTmG ES cells and found robust expression of eGFP in beating cardiomyocytes (Online Figure II).
As αMHC is a marker of mature cardiomyocytes, we hypothesized that overexpression of developmentally essential genes Gata4/Mef2c/Tbx5 might induce an immature cardiac phenotype. We overexpressed GMT factors in TTFs from Nkx2.5 knock-in Cre/ ROSA26mTmG reporter mice, which express eGFP in immature cardiomyocytes (Figure 1a). No eGFP+ cells were detected after three weeks among infected fibroblasts (Figure 1e), suggesting a lack of Nkx2.5 upregulation.
Reprogramming cardiac fibroblasts by GMT overexpression
We hypothesized that reprogramming CFs might be more efficient than reprogramming TTFs as CFs share developmental lineage history with cardiomyocytes. We overexpressed GMT in CFs from 2–3 week old αMHC-Cre/ROSA26mTmG mice and FACS-purified Thy1.2+/eGFP- CFs from Nkx2.5-Cre /ROSA26mTmG hearts. We first confirmed the upregulation of GMT in infected CFs (Online Figure Ie). Although Gata4 expression was increased by only 8-fold compared with uninfected CFs, we found a high baseline level of Gata4 expression in CFs (Online Figure If). Three weeks after GMT overexpression, we found no eGFP+ cells among either αMHC-Cre/ROSA26mTmG or Nkx2.5-Cre/ROSA26mTmG CFs (Figure 1f, g).
Quantitative PCR analysis of cardiac genes following GMT overexpression
As neither αMHC- nor Nkx2.5 lineage-reporting fibroblasts conveyed cardiac reprogramming, we suspected that not all cardiac genes are equally induced by GMT overexpression. Quantitative PCR analysis of GMT-infected TTFs across a panel of cardiac genes confirmed the induction of some but not all cardiac genes (Figure 1h). Interestingly, while cTnT levels post-infection appeared modest compared with the high levels found in E10.5 hearts (Figure 1h), this represented a 250-fold increase in cTnT expression compared with uninfected TTFs (Online Figure III). In GMT-infected CFs, transcript levels of SERCA2a, Tbx20, and Gata6 were comparable to those in E10.5 cardiomyocytes (Figure 1i). Levels of cTnT, MyBPC and Gja1 also significantly increased but a number of important sarcomeric proteins failed to be induced.
Since cTnT was robustly upregulated by GMT factors, we overexpressed GMT in freshly-isolated TTFs from cTnT-Cre/ROSA26mTmG mice. Remarkably, up to ~35% of the cells became eGFP+ three weeks post-infection (Figure 2a). However, we noted that eGFP+ cells remained morphologically indistinguishable from eGFP- cells and exhibited no spontaneous beating activity (Figure 2b).
Figure 2
Figure 2
Gene expression and functional analysis of GMT infected fibroblasts
Global transcriptional profiles of TTFs and CFs before and after GMT overexpression
To further examine GMT-induced changes to gene expression on a genome-wide scale, we performed microarrays of CFs and TTFs before and after GMT infection. We selected for Tbx5 expressing cells by using a Tbx5-IRES-Puro lentivirus and treating the GMT infected cells with puromycin. Interestingly, we found no significant change in global gene expression profiles of CFs and TTFs after GMT overexpression (Online Figure IV). We noted, however, a subset of cardiac genes shifted towards cardiomyocyte-like expression patterns but these genes were either experimentally introduced (e.g. Tbx5) or known from the qPCR data above (e.g. cTnT) (data not shown).
Electrophysiological assessment of GMT-overexpressing fibroblasts
While the global gene expression data shows a low overall efficiency of cardiomyocyte reprogramming by GMT factors, possibility remains that rare cells are more fully reprogrammed. To investigate this, we performed electrophysiological assessment of GMT infected TTFs at a single cell level. We compared GMT-overexpressing TTFs (n=32) with uninfected fibroblasts (n=26) and ES cell-derived cardiomyocytes (n=20) at three weeks post-infection. We found no spontaneous action potentials in GMT-infected (0/32 cells) or uninfected (0/26 cells) TTFs while ES cell-derived cardiomyocytes were all spontaneously active (20/20 cells) (data not shown). Upon pacing, ES cell-derived cardiomyocytes displayed typical murine cardiac action potentials, while uninfected fibroblasts demonstrated passive exponential decay of membrane potential consistent with a lack of active repolarization (26/26 cells) (Figure 2c). Interestingly, 7/32 (21.8%) of GMT-infected cells demonstrated up-sloping pacing induced action potential followed by passive exponential decay (Figure 2c).
We further examined this GMT-induced depolarization response in TTFs by introducing increasing stimulus amplitudes and found a graded response distinct from the “all or none” sodium current-dependent excitation typical of cardiomyocytes15 (Figure 2d). This absence of inward voltage-activated sodium currents in GMT-infected TTFs and their lack of active repolarization is likely responsible for their inability to fire repetitively upon high frequency pacing stimulation (data not shown). The ability of nifedipine, a dihydropyridine calcium channel antagonist, to block pacing induced action potentials (Figure 2e, red curve) revealed that the predominant component of these transient depolarizations was mediated by calcium and not sodium channels.
Survival and reprogramming of transplanted GMT-overexpressing fibroblasts
The low efficiency observed in GMT-overexpressing fibroblasts in vitro could have been explained by the absence of a supportive reprogramming environment. To examine the influence of a myocardial environment on reprogramming, we overexpressed GMT in CFs derived from transgenic mice that constitutively express luciferase and eGFP16 and injected these cells into the hearts of female SCID mice (5×105 cells/heart, n=3) that had just undergone surgical ligation of their left anterior descending (LAD) coronary arteries (Figure 3a). In parallel, uninfected cardiac-derived cells16 (5×105 cells/heart) were injected into the injured hearts of other SCID mice (n=3) as controls. Bioluminescence imaging over 8 days revealed a rapid loss of luciferase activity in hearts transplanted with GMT-infected CFs while only a modest degree of attrition was observed among uninfected cells (Figure 3b, c). To assess whether engrafted GMT overexpressing fibroblasts underwent cardiomyocyte reprogramming, we recovered transplanted single eGFP+ cells by FACS and evaluated their expression of a panel of cardiac genes using a novel Fluidigm® single cell PCR array. We found that recovered cells predominantly expressed vimentin, a marker of fibroblasts, while rare cells expressed a small number of cardiac genes (Figure 3d).
Figure 3
Figure 3
Survival and reprogramming of GMT infected fibroblasts in an experimental model of MI
Direct cardiomyocyte reprogramming by overexpression of cardiac transcription factors is a conceptually appealing strategy for cardiomyocyte regeneration. Using transgenic mice expressing Cre recombinase under the regulation of αMHC, Nkx2.5 and cTnT promoters, we found that GMT overexpression in TTFs and CFs only induced expression of a subset of cardiac genes with minimal alteration of the fibroblast phenotype. We detected calcium channel-mediated depolarization currents in a subset of infected cells, suggesting that GMT reprogramming factors induced incomplete electrophysiological reprogramming. Transplantation of GMT-infected CFs into injured hearts resulted in no further improvements in the efficiency of cardiomyocyte phenotype conversion. Altogether, these data support a need for improved efficiency in cardiomyocyte reprogramming. A greater understanding of epigenetic changes associated with transcription factor overexpression will enhance the therapeutic potential of this approach.
Recent reports of direct reprogramming of fibroblast into other tissues such blood progenitors and neurons by overexpression of lineage-specific transcription factors17,18 offer hope that we may apply similar strategies to cardiac regenerative therapies. In our hands, however, the overall efficiency of cardiomyocyte reprograming with GMT overexpression is extremely low. Potential differences in experimental protocols (e.g. the method of fibroblast isolation, the method of virus production) or reagents used (e.g. genetic background of mouse strain, the cardiomyocyte-lineage reporters used) can influence the level of GMT overexpression and may account for some of the differences between our findings and those of Ieda et al.10 As an example, we found significant differences in the interpretations of reprogramming efficiency when different reporters (e.g. cTnT vs αMHC or Nkx2.5) are used. It is worth mentioning that the percentage of cTnT expressing cells in Ieda et al was only ~5% of the total infected cell population and among these, only a fraction of them are likely to express a more complete cardiac gene expression.
Our results highlight many challenges in transcription factor-based cardiac reprogramming. Importantly, we demonstrated the profound influences that choices of lineage reporters, cell types, and methods of evaluating cardiac phenotypes have on assessments of reprogramming efficiency. Moreover, our study raises important caveats for using GMT-reprogrammed fibroblasts as transplantable cardiomyocyte-like cells because these cells demonstrate poor survival post-transplantation (a common finding in previous cardiac transplantation experiments6) and are therefore unlikely to integrate with surrounding cardiomyocytes. Whether by adding different transcription factors and epigenetic modifiers to the GMT mix or by changing the starting cell type, significant improvements in the efficiency of cardiomyocyte reprogramming are needed before this strategy can be applied therapeutically.
Novelty and Significance
What is known
  • Cellular reprogramming is a potentially useful strategy for generating therapeutically important cell types such as cardiomyocytes
  • Gata4, Mef2c, and Tbx5 (GMT) have been in vitroreported to reprogram fibroblasts into cardiomyocytes as well as in vivo.
What new information does this article contribute?
  • The efficiency of direct cardiac reprogramming by GMT overexpression in cardiac fibroblasts (CF) and tail tip fibroblasts (TTF) is very low.
  • While GMT overexpression upregulates a subset of cardiac genes and alters the electrophysiological phenotype in fibroblasts, this phenotype does not resemble those of a bona fide cardiomyocyte.
The replacement of lost cardiomyocytes using more abundant cell types is an important goal in cardiac regenerative medicine. This study examines the efficiency of direct fibroblast reprograming into cardiomyocytes by the overexpression of GMT. In contrast to previous reports, it is found that GMT overexpression is inefficient at inducing a mature cardiomyocyte phenotype in TTF and CF. Furthermore, transplantation of GMT overexpressing CFs into injured murine hearts resulted in poor cell survival and minimal expression of cardiac genes. This study demonstrates the ongoing challenges in our ability to efficiently reprogram fibroblasts into cardiomyocytes for therapeutic applications.
Supplementary Material
Acknowledgments
We thank Dr. William Pu at Boston Children’s Hospital for cTnt-Cre/ROSAmtmg reporter fibroblasts; Dr. Robert Schwartz at University of Houston for Nkx2.5-Cre knock-in mice; Drs. X.J. Yang and Qi Kenneth Wang at Cleveland Clinic and Case Western University for promoter constructs used in luciferase reporter assays; Drs. Konrad Hochedlinger and Matthias Stadtfeld for the rtTA plasmid vector. Microarray studies were performed by the Molecular Genetics Core Facility at Children’s Hospital Boston supported by NIH-P50-NS40828, and NIH-P30-HD18655. Ms. Laura Prickett-Rice at the MGH-Center for Regenerative Medicine flow cytometry core facility provided assistance with FACS.
Funding
M. K. was supported by the German Research Foundation (KR3770/1-1). M.-A.D. was supported by the German Heart Foundation (Deutsche Herzstiftung e.V.). D.J.M was supported by NIH grants DA026982 and HL109004. S.M.W. was supported by NHLBI (HL081086, HL100408), NIH/OD (004411) and the Harvard Stem Cell Institute.
Non-Standard Abbreviations
αMHCalpha myosin heavy chain
CFcardiac fibroblast
cTnTcardiac troponin T
eGFPenhanced green fluorescent protein
Eembryonic day
ESCembryonic stem cell(s)
Gata4GATA binding protein 4
Gja1gap junction protein, alpha 1
GMTGata4/Mef2c/Tbx5
MyBPCmyosin-binding protein C
Mef2cmyocyte-specific enhancer factor 2c
Nkx2.5NK2 homebox 5
SERCA2asarcoplasmic reticulum calcium ATPase 2a
Tbx5T-box 5
Tbx20T-box 20
TTFtail tip fibroblast

Footnotes
Disclosures
None.
1. Kikuchi K, Holdway JE, Werdich AA, Anderson RM, Fang Y, Egnaczyk GF, Evans T, MacRae CA, Stainier DYR, Poss KD. Primary contribution to zebrafish heart regeneration by gata4+ cardiomyocytes. Nature. 2010;464:601–605. [PMC free article] [PubMed]
2. Vilahur G, Juan-Babot O, Peña E, Onate B, Casaní L, Badimon L. Molecular and cellular mechanisms involved in cardiac remodeling after acute myocardial infarction. Journal of Molecular and Cellular Cardiology. 2011;50:522–533. [PubMed]
3. Kehat I, Khimovich L, Caspi O, Gepstein A, Shofti R, Arbel G, Huber I, Satin J, Itskovitz-Eldor J, Gepstein L. Electromechanical integration of cardiomyocytes derived from human embryonic stem cells. Nature Biotechnology. 2004;22:1282–1289. [PubMed]
4. Zimmermann W-H, Melnychenko I, Wasmeier G, Didie M, Naito H, Nixdorff U, Hess A, Budinsky L, Brune K, Michaelis B, Dhein S, Schwoerer A, Ehmke H, Eschenhagen T. Engineered heart tissue grafts improve systolic and diastolic function in infarcted rat hearts. Nature Medicine. 2006;12:452–458. [PubMed]
5. Furuta A, Miyoshi S, Itabashi Y, Shimizu T, Kira S, Hayakawa K, Nishiyama N, Tanimoto K, Hagiwara Y, Satoh T, Fukuda K, Okano T, Ogawa S. Pulsatile Cardiac Tissue Grafts Using a Novel Three-Dimensional Cell Sheet Manipulation Technique Functionally Integrates With the Host Heart, In Vivo. Circ Res. 2006;98:705–712. [PubMed]
6. Laflamme MA, Chen KY, Naumova AV, Muskheli V, Fugate JA, Dupras SK, Reinecke H, Xu C, Hassanipour M, Police S, O’Sullivan C, Collins L, Chen Y, Minami E, Gill EA, Ueno S, Yuan C, Gold J, Murry CE. Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nature Biotechnology. 2007;25:1015–1024. [PubMed]
7. van Laake LW, Passier R, Monshouwer-Kloots J, Verkleij AJ, Lips DJ, Freund C, den Ouden K, Ward-van Oostwaard D, Korving J, Tertoolen LG, van Echteld CJ, Doevendans PA, Mummery CL. Human embryonic stem cell-derived cardiomyocytes survive and mature in the mouse heart and transiently improve function after myocardial infarction. Stem Cell Research. 2007;1:9–24. [PubMed]
8. Caspi O, Huber I, Kehat I, Habib M, Arbel G, Gepstein A, Yankelson L, Aronson D, Beyar R, Gepstein L. Transplantation of Human Embryonic Stem Cell-Derived Cardiomyocytes Improves Myocardial Performance in Infarcted Rat Hearts. Journal of the American College of Cardiology. 2007;50:1884–1893. [PubMed]
9. Miura K, Okada Y, Aoi T, Okada A, Takahashi K, Okita K, Nakagawa M, Koyanagi M, Tanabe K, Ohnuki M, Ogawa D, Ikeda E, Okano H, Yamanaka S. Variation in the safety of induced pluripotent stem cell lines. Nature Biotechnology. 2009;27:743–745. [PubMed]
10. Ieda M, Fu J-D, Delgado-Olguin P, Vedantham V, Hayashi Y, Bruneau BG, Srivastava D. Direct Reprogramming of Fibroblasts into Functional Cardiomyocytes by Defined Factors. Cell. 2010;142:375–386. [PMC free article] [PubMed]
11. Muzumdar MD, Tasic B, Miyamichi K, Li L, Luo L. A global double-fluorescent Cre reporter mouse. genesis. 2007;45:593–605. [PubMed]
12. Brown CO, Chi X, Garcia-Gras E, Shirai M, Feng X-H, Schwartz RJ. The Cardiac Determination Factor, Nkx2–5, Is Activated by Mutual Cofactors GATA-4 and Smad1/4 via a Novel Upstream Enhancer. J Biol Chem. 2004;279:10659–10669. [PubMed]
13. Lien CL, Wu C, Mercer B, Webb R, Richardson JA, Olson EN. Control of early cardiac-specific transcription of Nkx2–5 by a GATA-dependent enhancer. Development. 1999;126:75–84. [PubMed]
14. Wang AH, Bertos NR, Vezmar M, Pelletier N, Crosato M, Heng HH, Th’ng J, Han J, Yang X-J. HDAC4, a Human Histone Deacetylase Related to Yeast HDA1, Is a Transcriptional Corepressor. Mol CellBiol. 1999;19:7816–7827. [PMC free article] [PubMed]
15. Kleber AG, Rudy Y. Basic Mechanisms of Cardiac Impulse Propagation and Associated Arrhythmias. Physiol Rev. 2004;84:431–488. [PubMed]
16. Li Z, Lee A, Huang M, Chun H, Chung J, Chu P, Hoyt G, Yang P, Rosenberg J, Robbins RC, Wu JC. Imaging Survival and Function of Transplanted Cardiac Resident Stem Cells. Journal of the American College of Cardiology. 2009;53:1229–1240. [PMC free article] [PubMed]
17. Szabo E, Rampalli S, Risueno RM, Schnerch A, Mitchell R, Fiebig-Comyn A, Levadoux-Martin M, Bhatia M. Direct conversion of human fibroblasts to multilineage blood progenitors. Nature. 2010;468:521–526. [PubMed]
18. Vierbuchen T, Ostermeier A, Pang ZP, Kokubu Y, Sudhof TC, Wernig M. Direct conversion of fibroblasts to functional neurons by defined factors. Nature. 2010;463:1035–1041. [PMC free article] [PubMed]